Multi-pathway macrophage pyroptosis mediates necrotizing enterocolitis: integrated sequencing and animal validation

Abstract

Abstract Background Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disorder prevalent in preterm infants, yet its core pathogenic mechanism remains poorly defined. Pyroptosis is a proinflammatory programmed cell death closely linked to intestinal injury, but whether multiple pyroptotic pathways act synergistically and which cell type dominates pyroptotic activation in NEC remain unclear. This study aimed to clarify the macrophage-centered multi-pathway pyroptosis network driving NEC intestinal damage and explore the therapeutic value of targeting the key pyroptosis effector GSDMD. Methods We integrated human clinical intestinal transcriptome data (single-cell and bulk RNA sequencing) and a neonatal mouse NEC model. Consensus clustering stratified NEC patients into high- and low-pyroptosis subtypes. SHAP algorithm identified core pyroptosis regulators. Western blot and ELISA detected pyroptotic proteins and inflammatory cytokines in mouse intestines; disulfiram was administered to verify the protective effect of GSDMD inhibition. Results Macrophages were confirmed as the primary cell subset with elevated pyroptotic signatures. Canonical, noncanonical, and CASP8-axis pyroptotic pathways were synchronously activated in human NEC tissues, with the canonical pathway dominant and IFI16 the top susceptibility gene. High-pyroptosis NEC lesions exhibited concurrent enrichment of proinflammatory myeloid cells and regulatory T cells. In mice, elevated cleaved GSDMD and mature IL-1β aggravated intestinal lesions, while disulfiram-mediated GSDMD suppression alleviated pathological injury. Conclusions Macrophage-driven synergistic activation of three pyroptotic cascades constitutes the central pathological driver of NEC. GSDMD represents a promising therapeutic target, and pyroptosis-based molecular subtyping provides a novel strategy for stratified clinical intervention of NEC.